Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes

¹ Unidad de Biofísica
² Humboldt Universität zu Berlin

^* To whom correspondence should be addressed. E-mail: gbpgourf{at}lg.ehu.es.

Submitted on July 29, 2004
Revised on August 23, 2004
Accepted on 15 September 2004

	Abstract

Transbilayer lipid motion in membranes may be important in certain physiological events, such as ceramide signalling. In the present study, the transbilayer redistribution of lipids induced either by ceramide addition or by enzymatic ceramide generation at one side of the membrane has been monitored using pyrene-labeled phospholipid analogues. When added in organic solution to preformed liposomes, egg ceramide induced transbilayer lipid motion in a dose-dependent way. Short-chain (C6 and C2) ceramides were less active than egg ceramide, while dihydroceramides or dioleoylglycerol were virtually inactive in promoting flip-flop. The same results were obtained when ceramides, dihydroceramides or diacylglycerols were generated in situ through the action of a sphingomyelinase, or of a phospholipase C. The phenomenon was dependent on the bilayer lipid composition, being faster in the presence of lipids that promote inverted phase formation, e.g. phosphatidylethanolamine and cholesterol, and, conversely, slower in the presence of lysophosphatidylcholine, that inhibits inverted phase formation. Transbilayer motion was almost undetectable in bilayers composed of pure phosphatidylcholine, or pure sphingomyelin. The use of pyrene-phosphatidylserine allowed detection of flip-flop movement induced by egg ceramide in human red blood cell membranes at a rate comparable to that observed in model membranes. The data suggest that when one membrane leaflet becomes enriched in ceramides, they diffuse towards the other leaflet. This is counterbalanced by lipid movement in the opposite direction, so that net mass transfer between monolayers is avoided. These observations may be relevant to the physiological mechanism of transmembrane signalling via ceramides.

Key Words: cell signalling, ceramides, flip-flop, pyrene excimer fluorescence, sphingomyelinase, transbilayer lipid motion

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